J Nutrigenet Nutrigenomics 2014;7:264–273 DOI: 10.1159/000381676 Received: November 24, 2014 Accepted: March 14, 2015 Published online: May 8, 2015
© 2015 S. Karger AG, Basel 1661–6499/15/0076–0264$39.50/0 www.karger.com/jnn
Original Paper
Vitamin D Receptor Genotype Modulates the Correlation between Vitamin D and Circulating Levels of let-7a/b and Vitamin D Intake in an Elderly Cohort Emma Louise Beckett a, d Charlotte Martin a Konsta Duesing d Patrice Jones a John Furst b Zoe Yates c Martin Veysey e Mark Lucock a a
School of Environmental and Life Sciences, b School of Mathematical and Physical Sciences, and c School of Biomedical Sciences and Pharmacy, University of Newcastle, Ourimbah, N.S.W., d Food and Nutrition Flagship, CSIRO, North Ryde, N.S.W., and e Teaching and Research Unit, Central Coast Local Health District, Gosford, N.S.W., Australia
Key Words let-7 microRNA · Vitamin D · Vitamin D receptor · BsmI restriction site · ApaI restriction site · Nutrigenomics Abstract Background and Aims: Circulating microRNAs (miRNAs) are linked to disease and are potential biomarkers. Vitamin D may modulate miRNA profiles, and vitamin D status has been linked to risk of disease, including cardiovascular disease and cancers. We hypothesise that genotypic variance influences these relationships. We examined the correlations between vitamin D intake and circulating levels of the miRNAs let-7a/b, and the involvement of two common vitamin D receptor (VDR) polymorphisms, BsmI and ApaI. Methods: Two hundred participants completed food frequency and supplement questionnaires, and were assayed for circulating let-7b expression by qPCR. Polymorphisms were detected using restriction fragment length polymorphism-PCR. Results: let-7b expression negatively correlated with vitamin D intake (rs = –0.20, p = 0.005). The magnitude and direction of correlation were maintained in the presence of the BsmI restriction site (rs = –0.27, p = 0.0005). However, in the absence of BsmI restriction site, the direction of the correlation was reversed (rs = +0.319, p = 0.0497). These correlations were significantly different (z-score = 2.64, p = 0.0085). The correlation between
Emma Louise Beckett School of Environmental and Life Sciences University of Newcastle Brush Rd, Ourimbah, NSW 2258 (Australia) E-Mail emma.beckett @ uon.edu.au
Downloaded by: Kellogg Health Sciences Libr. 129.173.72.87 - 5/18/2015 5:34:52 AM
This paper was presented at the 8th Congress of the International Society of Nutrigenetics/Nutrigenomics (ISNN), Gold Coast, Qld., Australia, May 2–3, 2014.
265
J Nutrigenet Nutrigenomics 2014;7:264–273 DOI: 10.1159/000381676
© 2015 S. Karger AG, Basel www.karger.com/jnn
Beckett et al.: Vitamin D Receptor Genotype Modulates the Correlation between Vitamin D and Circulating Levels of let-7a/b and Vitamin D Intake in an Elderly Cohort
vitamin D intake and let-7a was only significant in those without the ApaI restriction site. Conclusions: The correlation between vitamin D intake and let-7a/b expression in this cohort varies with VDR genotype. This study highlights the importance of considering underlying genotypic variance in miRNA expression studies and in nutritional epigenetics generally. © 2015 S. Karger AG, Basel
MicroRNAs (miRNAs) are a class of short non-coding RNAs that can regulate gene expression at the post-transcriptional level, normally by blocking mRNA translation or triggering degradation [1–5]. miRNAs are estimated to participate in nearly all cellular processes [6]. To date, over 2,500 mature miRNAs have been identified (miRBase, http://www.mirbase. org, release June 21, 2014). Disease-specific miRNA profiles have been identified, including for those diseases with known dietary risk factors [7–9]. Evidence is mounting that miRNA profiles can be modulated by nutrients and bioactive compounds [10]. Traditionally, the focus has been on tissue expression of miRNAs, but circulating miRNAs have recently been identified in serum and plasma, which have the advantage of being easily accessible for use as biomarkers. These circulating miRNAs are now being linked to particular diseases [11]. let-7a and let-7b are well-characterised members of the let-7 family of tumour suppressor miRNAs. They are highly expressed in the cardiovascular system [12] and are robustly detected in plasma samples [13–15]. Altered expression of let-7 has been implicated in a number of later-life diseases; circulating let-7b has been suggested as a potential biomarker for cardiovascular diseases, such as atherosclerosis and myocardial infarction [12], and the let-7 family may be involved in regulation of glucose homeostasis in diabetes [16]. Furthermore, expression of let-7a/b is often reduced in malignant tissue, relative to healthy tissue [17], and in some cases these altered profiles are reflected in blood circulation [14, 18]. For circulating miRNAs to be valuable as biomarkers for disease, it is important to understand how other environmental factors influence their expression levels. Vitamin D is well known for the influence it has on gene expression. The active vitamin D metabolite 1,25-dihydroxyvitamin D3 (calcitriol) directly regulates gene transcription via the vitamin D receptor (VDR), which acts as a nuclear receptor transcription factor [19]. The potential for vitamin D to modulate gene expression via additional indirect pathways is now being investigated. Vitamin D is synthesised in the skin in response to sunlight, consumed in the diet as cholecalciferol (vitamin D3) or ergocalciferol (vitamin D2), or taken as a supplement. Although current recommended intakes are based only on its role in bone health [20], epidemiological evidence is now mounting for a potential role for suboptimal vitamin D levels in multiple conditions, including diabetes [21], cardiovascular disease [22], autoimmune disease [23] and cancers [24]; however, results to date remain inconsistent [20]. Vitamin D supplementation is now being investigated in the treatment and prevention of a number of these conditions [25–27], in particular its potential as a cancer chemopreventative, due to the identified role of the VDR in cell cycle regulation and differentiation [28]. Multiple in vitro models using malignant cell lines have demonstrated the anti-proliferative effect of vitamin D [29–32], which correlates with the altered expression of a number of miRNAs, including the let-7 family [33–36], suggesting that aberrant expression of these miRNAs may contribute to the malignant phenotype and that this can be moderated by vitamin D treatment [37]. In a model of serum starvation using a breast epithelial cell line (MCF12F), calcitriol treatment had a protective effect. Serum starvation led to a significant increase in the expression of multiple miRNAs, including the let-7 family; however, this was reversed in the presence of calcitriol, suggesting miRNA expression may be a mechanism
Downloaded by: Kellogg Health Sciences Libr. 129.173.72.87 - 5/18/2015 5:34:52 AM
Introduction
266
J Nutrigenet Nutrigenomics 2014;7:264–273 DOI: 10.1159/000381676
© 2015 S. Karger AG, Basel www.karger.com/jnn
Beckett et al.: Vitamin D Receptor Genotype Modulates the Correlation between Vitamin D and Circulating Levels of let-7a/b and Vitamin D Intake in an Elderly Cohort
protecting against cellular stress via a vitamin D-related process [38], which may have implications in multiple disease states including diabetes, cardiovascular disease, and cancers [39]. Limited studies investigating the correlation between serum levels of vitamin D and miRNA expression in humans have been conducted. In a study of 13 pregnant women, categorised by low (≤25.5 ng/ml) or high (≥31.7 ng/ml) plasma calcitriol level, 11 miRNAs were differentially regulated [40]. In a small study, 2 groups of 5 subjects were given high doses of vitamin D for 12 months, and their miRNA levels were assessed relative to baseline. Several miRNAs were differentially expressed after 12 months, including let-7a [41], an established target of VDR, with a vitamin D response element (VDRE) in the regulatory region of the gene controlling its expression [42]. To further investigate the role of vitamin D in modulating levels of circulating miRNAs, we studied the relationship between vitamin D intake and the expression of circulating let-7a/b, 2 well-characterised tumour-suppressor miRNAs, in a large human cohort. To further investigate the role of the VDR in this relationship, two common polymorphisms in the VDR gene, BsmI (rs1544410) and ApaI (rs7975232), were assayed in participants. Methods Subjects and Sample Collection This study consisted of 200 participants, aged 65 years or more, living on the Central Coast of New South Wales, Australia, who completed a food frequency and supplement questionnaire, and donated blood for analysis. Blood was collected in heparin-lined tubes (Greiner Bio-one, Frickenhausen, Germany), and plasma was isolated by centrifugation. Informed consent was obtained prior to participation under University of Newcastle Human Research Ethics Committee approval number H-2008-0431. Food Frequency and Supplement Questionnaires Daily intake of vitamin D was estimated using a validated food frequency questionnaire, covering 225 food items and all food groups. Subjects also provided a list of all supplements and their frequency of intake. The food frequency questionnaires were analysed using FoodworksTM (version 2.10.146; Xyris Software, Brisbane, Qld., Australia) [43, 44]. The correlation between estimated intake and plasma concentrations of 25(OH) vitamin D has been validated in a subset (n = 80; adjusted r2 = 0.46, p < 0.001) of this cohort (see online suppl. methods and fig. S1; see www.karger.com/doi/10.1159/000381676 for all online suppl. material). Genotyping Genomic DNA was isolated from whole blood and amplified using PCR. Restriction fragment length polymorphism assays and gel electrophoresis were used to genotype two diallelic polymorphisms of the VDR gene, BsmI G/A (rs1544410) and ApaI A/C (rs7975232) [43, 45], both located on the last intron [46]. The presence of restriction sites for the BsmI and ApaI enzymes were coded as ‘b’ and ‘a’ and the absence of restriction sites as ‘B’ and ‘A’, respectively (see online suppl. methods for additional details).
Statistics Statistical analyses were performed using JMP (version 11; SAS Institute Inc., Cary, N.C., USA). Correlations between continuous variables were assessed using Spearman’s correlation coefficient due to the nonnormal distribution of vitamin D intake in this cohort; z-scores were used to assess significant differences
Downloaded by: Kellogg Health Sciences Libr. 129.173.72.87 - 5/18/2015 5:34:52 AM
Circulating let-7a/b Expression by qPCR miRNAs were isolated from plasma using TRIzol LS and GlycoBlue [47]. Three synthetic Caenorhabditis elegans miRNAs (cel-238, 54, 38) were added to plasma samples as exogenous spike in controls to normalise data as no appropriate endogenous housekeeping gene exists in plasma [48]. cDNA was synthesised using universal primers, and qPCR primer design was conducted as per the rules described in Balcells et al. [49].
267
J Nutrigenet Nutrigenomics 2014;7:264–273 DOI: 10.1159/000381676
© 2015 S. Karger AG, Basel www.karger.com/jnn
Beckett et al.: Vitamin D Receptor Genotype Modulates the Correlation between Vitamin D and Circulating Levels of let-7a/b and Vitamin D Intake in an Elderly Cohort
Table 1. Allelic and genotypic frequencies of VDR SNPs BsmI (rs1544410) and ApaI (rs7975232)
rs1544410 B allele b allele BB genotype Bb genotype bb genotype rs7975232 A allele a allele AA genotype Aa genotype aa genotype
38.7% 61.2% 12.0% 53.5% 34.5% 51.0% 49.0% 27.0% 48.0% 25.0%
Upper case signifies restriction site absent, lower case signifies restriction site present.
between correlations (http://vassarstats.net/rdiff.html). Analyses were performed correcting for age and sex; however, this had no notable significant impact on results. Categorical data were assessed using the Mann-Whitney test when comparing two categories, and the Kruskal-Wallis test was used when comparing multiple categories. Differences between groups were considered to be statistically significant at p ≤ 0.05.
Results
Participant Characteristics and Vitamin D Intake The mean age of participants was 75.0 ± 0.5 years (range: 65–94 years), and 57.5% were female. The median daily reported vitamin D intake was 6.8 μg/day (range: 0–65.6 μg/day). Fifty-seven (28.5%) participants reported taking a supplement containing vitamin D. The recommended adequate daily intake for vitamin D in Australia is 10 μg/day for 51- to 70-yearolds and 15 μg/day for those aged over 70. The recommended upper limit of consumption is 80 μg/day [50]. Based on these criteria, 177 participants had inadequate intake for their age group, and none had excess intake above the upper limit.
Association between Vitamin D Intake and let-7a/b Expression A weak but significant negative association was found between let-7b expression and vitamin D intake (table 2; rs = –0.20, p = 0.005). The same trend was seen for let-7a, but this did not reach statistical significance (table 2; rs = –0.14, p = 0.057). Adjustment for age and sex did not significantly alter these relationships. Categorical analysis was conducted on the data, based on adequate daily intake (greater than or adequate daily intake vs. less than adequate daily intake), as these are clinically relevant categories. There was a significant difference between the mean let-7b expression
Downloaded by: Kellogg Health Sciences Libr. 129.173.72.87 - 5/18/2015 5:34:52 AM
VDR Genetic Variants The allelic and genotypic frequencies for both polymorphisms tested are given in table 1. The genotype frequency did not deviate from Hardy-Weinberg equilibrium expectations for either the BsmI or the ApaI polymorphism (p = 0.19 and 0.85, respectively). These two polymorphisms exhibit strong linkage disequilibrium [51]. In this population, 91.7% of those with the BB genotype (BsmI restriction site absent) were also AA (ApaI restriction site absent); however, the AA genotype was more than twice as frequent as the BB genotype.
268
J Nutrigenet Nutrigenomics 2014;7:264–273 © 2015 S. Karger AG, Basel www.karger.com/jnn
DOI: 10.1159/000381676
Beckett et al.: Vitamin D Receptor Genotype Modulates the Correlation between Vitamin D and Circulating Levels of let-7a/b and Vitamin D Intake in an Elderly Cohort
Fig. 1. let-7b (a) and let-7a (b) expression in those who reported consuming adequate versus inadequate levels of vitamin D (n = 200). Data are presented as means and standard error of means. ns = Not significant. * p < 0.05.
a
b
Table 2. Correlation between vitamin D intake and let-7a/b expression, stratified by absence/presence of BsmI (rs1544410) and ApaI (rs7975232) restriction polymorphism Total (n = 200)
BsmI
z-score
let-7b
–0.20 (0.005)
–0.27 (0.0005)
let-7a
–0.1367 (0.0566) –0.0655 (0.3958) 0.0254 (0.9106)
restriction site restriction site present (n = 176) absent (n = 24) 0.319 (0.049)
ApaI
z-score
restriction site restriction site present (n = 146) absent (n = 54) 2.64 (0.0085)
–0.2899 (0.0006) 0.0614 (0.6621)
2.21 (0.027)
1.14 (0.12)
–0.0209 (0.086)
2.12 (0.034)
0.3132 (0.0224)
Figures indicate rs (p) values. Italic type highlights those relationships with a p value
© 2015 S. Karger AG, Basel 1661–6499/15/0076–0264$39.50/0 www.karger.com/jnn
Original Paper
Vitamin D Receptor Genotype Modulates the Correlation between Vitamin D and Circulating Levels of let-7a/b and Vitamin D Intake in an Elderly Cohort Emma Louise Beckett a, d Charlotte Martin a Konsta Duesing d Patrice Jones a John Furst b Zoe Yates c Martin Veysey e Mark Lucock a a
School of Environmental and Life Sciences, b School of Mathematical and Physical Sciences, and c School of Biomedical Sciences and Pharmacy, University of Newcastle, Ourimbah, N.S.W., d Food and Nutrition Flagship, CSIRO, North Ryde, N.S.W., and e Teaching and Research Unit, Central Coast Local Health District, Gosford, N.S.W., Australia
Key Words let-7 microRNA · Vitamin D · Vitamin D receptor · BsmI restriction site · ApaI restriction site · Nutrigenomics Abstract Background and Aims: Circulating microRNAs (miRNAs) are linked to disease and are potential biomarkers. Vitamin D may modulate miRNA profiles, and vitamin D status has been linked to risk of disease, including cardiovascular disease and cancers. We hypothesise that genotypic variance influences these relationships. We examined the correlations between vitamin D intake and circulating levels of the miRNAs let-7a/b, and the involvement of two common vitamin D receptor (VDR) polymorphisms, BsmI and ApaI. Methods: Two hundred participants completed food frequency and supplement questionnaires, and were assayed for circulating let-7b expression by qPCR. Polymorphisms were detected using restriction fragment length polymorphism-PCR. Results: let-7b expression negatively correlated with vitamin D intake (rs = –0.20, p = 0.005). The magnitude and direction of correlation were maintained in the presence of the BsmI restriction site (rs = –0.27, p = 0.0005). However, in the absence of BsmI restriction site, the direction of the correlation was reversed (rs = +0.319, p = 0.0497). These correlations were significantly different (z-score = 2.64, p = 0.0085). The correlation between
Emma Louise Beckett School of Environmental and Life Sciences University of Newcastle Brush Rd, Ourimbah, NSW 2258 (Australia) E-Mail emma.beckett @ uon.edu.au
Downloaded by: Kellogg Health Sciences Libr. 129.173.72.87 - 5/18/2015 5:34:52 AM
This paper was presented at the 8th Congress of the International Society of Nutrigenetics/Nutrigenomics (ISNN), Gold Coast, Qld., Australia, May 2–3, 2014.
265
J Nutrigenet Nutrigenomics 2014;7:264–273 DOI: 10.1159/000381676
© 2015 S. Karger AG, Basel www.karger.com/jnn
Beckett et al.: Vitamin D Receptor Genotype Modulates the Correlation between Vitamin D and Circulating Levels of let-7a/b and Vitamin D Intake in an Elderly Cohort
vitamin D intake and let-7a was only significant in those without the ApaI restriction site. Conclusions: The correlation between vitamin D intake and let-7a/b expression in this cohort varies with VDR genotype. This study highlights the importance of considering underlying genotypic variance in miRNA expression studies and in nutritional epigenetics generally. © 2015 S. Karger AG, Basel
MicroRNAs (miRNAs) are a class of short non-coding RNAs that can regulate gene expression at the post-transcriptional level, normally by blocking mRNA translation or triggering degradation [1–5]. miRNAs are estimated to participate in nearly all cellular processes [6]. To date, over 2,500 mature miRNAs have been identified (miRBase, http://www.mirbase. org, release June 21, 2014). Disease-specific miRNA profiles have been identified, including for those diseases with known dietary risk factors [7–9]. Evidence is mounting that miRNA profiles can be modulated by nutrients and bioactive compounds [10]. Traditionally, the focus has been on tissue expression of miRNAs, but circulating miRNAs have recently been identified in serum and plasma, which have the advantage of being easily accessible for use as biomarkers. These circulating miRNAs are now being linked to particular diseases [11]. let-7a and let-7b are well-characterised members of the let-7 family of tumour suppressor miRNAs. They are highly expressed in the cardiovascular system [12] and are robustly detected in plasma samples [13–15]. Altered expression of let-7 has been implicated in a number of later-life diseases; circulating let-7b has been suggested as a potential biomarker for cardiovascular diseases, such as atherosclerosis and myocardial infarction [12], and the let-7 family may be involved in regulation of glucose homeostasis in diabetes [16]. Furthermore, expression of let-7a/b is often reduced in malignant tissue, relative to healthy tissue [17], and in some cases these altered profiles are reflected in blood circulation [14, 18]. For circulating miRNAs to be valuable as biomarkers for disease, it is important to understand how other environmental factors influence their expression levels. Vitamin D is well known for the influence it has on gene expression. The active vitamin D metabolite 1,25-dihydroxyvitamin D3 (calcitriol) directly regulates gene transcription via the vitamin D receptor (VDR), which acts as a nuclear receptor transcription factor [19]. The potential for vitamin D to modulate gene expression via additional indirect pathways is now being investigated. Vitamin D is synthesised in the skin in response to sunlight, consumed in the diet as cholecalciferol (vitamin D3) or ergocalciferol (vitamin D2), or taken as a supplement. Although current recommended intakes are based only on its role in bone health [20], epidemiological evidence is now mounting for a potential role for suboptimal vitamin D levels in multiple conditions, including diabetes [21], cardiovascular disease [22], autoimmune disease [23] and cancers [24]; however, results to date remain inconsistent [20]. Vitamin D supplementation is now being investigated in the treatment and prevention of a number of these conditions [25–27], in particular its potential as a cancer chemopreventative, due to the identified role of the VDR in cell cycle regulation and differentiation [28]. Multiple in vitro models using malignant cell lines have demonstrated the anti-proliferative effect of vitamin D [29–32], which correlates with the altered expression of a number of miRNAs, including the let-7 family [33–36], suggesting that aberrant expression of these miRNAs may contribute to the malignant phenotype and that this can be moderated by vitamin D treatment [37]. In a model of serum starvation using a breast epithelial cell line (MCF12F), calcitriol treatment had a protective effect. Serum starvation led to a significant increase in the expression of multiple miRNAs, including the let-7 family; however, this was reversed in the presence of calcitriol, suggesting miRNA expression may be a mechanism
Downloaded by: Kellogg Health Sciences Libr. 129.173.72.87 - 5/18/2015 5:34:52 AM
Introduction
266
J Nutrigenet Nutrigenomics 2014;7:264–273 DOI: 10.1159/000381676
© 2015 S. Karger AG, Basel www.karger.com/jnn
Beckett et al.: Vitamin D Receptor Genotype Modulates the Correlation between Vitamin D and Circulating Levels of let-7a/b and Vitamin D Intake in an Elderly Cohort
protecting against cellular stress via a vitamin D-related process [38], which may have implications in multiple disease states including diabetes, cardiovascular disease, and cancers [39]. Limited studies investigating the correlation between serum levels of vitamin D and miRNA expression in humans have been conducted. In a study of 13 pregnant women, categorised by low (≤25.5 ng/ml) or high (≥31.7 ng/ml) plasma calcitriol level, 11 miRNAs were differentially regulated [40]. In a small study, 2 groups of 5 subjects were given high doses of vitamin D for 12 months, and their miRNA levels were assessed relative to baseline. Several miRNAs were differentially expressed after 12 months, including let-7a [41], an established target of VDR, with a vitamin D response element (VDRE) in the regulatory region of the gene controlling its expression [42]. To further investigate the role of vitamin D in modulating levels of circulating miRNAs, we studied the relationship between vitamin D intake and the expression of circulating let-7a/b, 2 well-characterised tumour-suppressor miRNAs, in a large human cohort. To further investigate the role of the VDR in this relationship, two common polymorphisms in the VDR gene, BsmI (rs1544410) and ApaI (rs7975232), were assayed in participants. Methods Subjects and Sample Collection This study consisted of 200 participants, aged 65 years or more, living on the Central Coast of New South Wales, Australia, who completed a food frequency and supplement questionnaire, and donated blood for analysis. Blood was collected in heparin-lined tubes (Greiner Bio-one, Frickenhausen, Germany), and plasma was isolated by centrifugation. Informed consent was obtained prior to participation under University of Newcastle Human Research Ethics Committee approval number H-2008-0431. Food Frequency and Supplement Questionnaires Daily intake of vitamin D was estimated using a validated food frequency questionnaire, covering 225 food items and all food groups. Subjects also provided a list of all supplements and their frequency of intake. The food frequency questionnaires were analysed using FoodworksTM (version 2.10.146; Xyris Software, Brisbane, Qld., Australia) [43, 44]. The correlation between estimated intake and plasma concentrations of 25(OH) vitamin D has been validated in a subset (n = 80; adjusted r2 = 0.46, p < 0.001) of this cohort (see online suppl. methods and fig. S1; see www.karger.com/doi/10.1159/000381676 for all online suppl. material). Genotyping Genomic DNA was isolated from whole blood and amplified using PCR. Restriction fragment length polymorphism assays and gel electrophoresis were used to genotype two diallelic polymorphisms of the VDR gene, BsmI G/A (rs1544410) and ApaI A/C (rs7975232) [43, 45], both located on the last intron [46]. The presence of restriction sites for the BsmI and ApaI enzymes were coded as ‘b’ and ‘a’ and the absence of restriction sites as ‘B’ and ‘A’, respectively (see online suppl. methods for additional details).
Statistics Statistical analyses were performed using JMP (version 11; SAS Institute Inc., Cary, N.C., USA). Correlations between continuous variables were assessed using Spearman’s correlation coefficient due to the nonnormal distribution of vitamin D intake in this cohort; z-scores were used to assess significant differences
Downloaded by: Kellogg Health Sciences Libr. 129.173.72.87 - 5/18/2015 5:34:52 AM
Circulating let-7a/b Expression by qPCR miRNAs were isolated from plasma using TRIzol LS and GlycoBlue [47]. Three synthetic Caenorhabditis elegans miRNAs (cel-238, 54, 38) were added to plasma samples as exogenous spike in controls to normalise data as no appropriate endogenous housekeeping gene exists in plasma [48]. cDNA was synthesised using universal primers, and qPCR primer design was conducted as per the rules described in Balcells et al. [49].
267
J Nutrigenet Nutrigenomics 2014;7:264–273 DOI: 10.1159/000381676
© 2015 S. Karger AG, Basel www.karger.com/jnn
Beckett et al.: Vitamin D Receptor Genotype Modulates the Correlation between Vitamin D and Circulating Levels of let-7a/b and Vitamin D Intake in an Elderly Cohort
Table 1. Allelic and genotypic frequencies of VDR SNPs BsmI (rs1544410) and ApaI (rs7975232)
rs1544410 B allele b allele BB genotype Bb genotype bb genotype rs7975232 A allele a allele AA genotype Aa genotype aa genotype
38.7% 61.2% 12.0% 53.5% 34.5% 51.0% 49.0% 27.0% 48.0% 25.0%
Upper case signifies restriction site absent, lower case signifies restriction site present.
between correlations (http://vassarstats.net/rdiff.html). Analyses were performed correcting for age and sex; however, this had no notable significant impact on results. Categorical data were assessed using the Mann-Whitney test when comparing two categories, and the Kruskal-Wallis test was used when comparing multiple categories. Differences between groups were considered to be statistically significant at p ≤ 0.05.
Results
Participant Characteristics and Vitamin D Intake The mean age of participants was 75.0 ± 0.5 years (range: 65–94 years), and 57.5% were female. The median daily reported vitamin D intake was 6.8 μg/day (range: 0–65.6 μg/day). Fifty-seven (28.5%) participants reported taking a supplement containing vitamin D. The recommended adequate daily intake for vitamin D in Australia is 10 μg/day for 51- to 70-yearolds and 15 μg/day for those aged over 70. The recommended upper limit of consumption is 80 μg/day [50]. Based on these criteria, 177 participants had inadequate intake for their age group, and none had excess intake above the upper limit.
Association between Vitamin D Intake and let-7a/b Expression A weak but significant negative association was found between let-7b expression and vitamin D intake (table 2; rs = –0.20, p = 0.005). The same trend was seen for let-7a, but this did not reach statistical significance (table 2; rs = –0.14, p = 0.057). Adjustment for age and sex did not significantly alter these relationships. Categorical analysis was conducted on the data, based on adequate daily intake (greater than or adequate daily intake vs. less than adequate daily intake), as these are clinically relevant categories. There was a significant difference between the mean let-7b expression
Downloaded by: Kellogg Health Sciences Libr. 129.173.72.87 - 5/18/2015 5:34:52 AM
VDR Genetic Variants The allelic and genotypic frequencies for both polymorphisms tested are given in table 1. The genotype frequency did not deviate from Hardy-Weinberg equilibrium expectations for either the BsmI or the ApaI polymorphism (p = 0.19 and 0.85, respectively). These two polymorphisms exhibit strong linkage disequilibrium [51]. In this population, 91.7% of those with the BB genotype (BsmI restriction site absent) were also AA (ApaI restriction site absent); however, the AA genotype was more than twice as frequent as the BB genotype.
268
J Nutrigenet Nutrigenomics 2014;7:264–273 © 2015 S. Karger AG, Basel www.karger.com/jnn
DOI: 10.1159/000381676
Beckett et al.: Vitamin D Receptor Genotype Modulates the Correlation between Vitamin D and Circulating Levels of let-7a/b and Vitamin D Intake in an Elderly Cohort
Fig. 1. let-7b (a) and let-7a (b) expression in those who reported consuming adequate versus inadequate levels of vitamin D (n = 200). Data are presented as means and standard error of means. ns = Not significant. * p < 0.05.
a
b
Table 2. Correlation between vitamin D intake and let-7a/b expression, stratified by absence/presence of BsmI (rs1544410) and ApaI (rs7975232) restriction polymorphism Total (n = 200)
BsmI
z-score
let-7b
–0.20 (0.005)
–0.27 (0.0005)
let-7a
–0.1367 (0.0566) –0.0655 (0.3958) 0.0254 (0.9106)
restriction site restriction site present (n = 176) absent (n = 24) 0.319 (0.049)
ApaI
z-score
restriction site restriction site present (n = 146) absent (n = 54) 2.64 (0.0085)
–0.2899 (0.0006) 0.0614 (0.6621)
2.21 (0.027)
1.14 (0.12)
–0.0209 (0.086)
2.12 (0.034)
0.3132 (0.0224)
Figures indicate rs (p) values. Italic type highlights those relationships with a p value
